Array-Based Analysis of Secreted Glycoproteins for Rapid Selection of a Single Cell Producing a Glycoprotein with Desired Glycosylation

Abstract

The therapeutic efficacy and in vivo biological function of a glycoprotein is significantly affected by its glycosylation profile. For the development of glycoproteins with therapeutic applications, selection of cell lines producing a glycoprotein with adequate glycoform is crucial. Here, we demonstrate an array-based analysis of secreted glycoproteins for rapid and efficient selection of a single cell producing a glycoprotein with desirable glycosylation. Our approach relies on microengraving and interrogation of glycoproteins produced by individual cells in a microwell array in terms of glycosylation profile as well as the produced amount. On the basis of statistical analysis of the interrogation, single cells which are predicted to produce a desired glycoprotein are selected, retrieved, and expanded. We applied the approach to human recombinant erythropoietin (rhEPO)-producing CHO cells and verified the selection of a single CHO cell that produces rhEPO with a high sialylation degree. Human erythropoietin (hEPO) bearing highly sialylated oligosaccharide was shown to display a much longer plasma half-life, resulting in high therapeutic efficacy. This method may find widespread use in the clonal selection for the production of other glycoproteins with specific glycosylation as well as analysis of the heterogeneity in cell populations in a high-throughput manner.

An 'omics approach towards CHO cell engineering

“…More recently, these techniques have been combined with automation to improve throughput and reduce the labor involved (Kacmar and Srienc, 2005; Shi et al, 2011). Recent advances in clonal selection based on quality of the bioengineered protein include high‐throughput analysis based on sialic acid content in the bioengineered glycoprotein (Markely et al, 2010; Park et al, 2010). The number of molecules of sialic acid attached to a bioengineered glycoprotein often impacts its biological activity (Kaneko et al, 2006), protease degradation (Goldwasser et al, 1974; Tsuda et al, 1990), serum half‐life (Ngantung et al, 2006), solubility (Sinclair and Elliott, 2005), and thermal denaturation (Goldwasser et al, 1974; Tsuda et al, 1990).…”

Single-cell assays

“…17 Kim et al used a similar approach to quantify the amount of sialylated oligosaccharide on secreted proteins. 18 The selection of glycoproteins with a specific glycosylation profile and the cells that produce them avoids the need for high-throughput analysis of large numbers of proteins.…”

A high-throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation

“…To monitor glycoprotein sialylation throughout the cell culture process, we need a high‐throughput method that can quickly analyze glycoprotein sialylation. Previous studies have developed high‐throughput methods for quantifying total molecule of sialic acid/molecule of glycoprotein . This information alone is, however, not sufficient to monitor the consistency of glycoprotein sialylation because the same glycoprotein with the same total molecule of sialic acid/molecule of glycoprotein may have different types of sialylated glycans that can affect product efficacy and safety.…”